The Kell blood group system is a well-known but complex group of blood antigens, comprising over 20 different related antigens. Of these, the antigen K1 (K, Kell) is known to be the strongest immunogen among the 23 known phenotypes. Serologically, the K1 sublocus has an allelic relationship with a high frequency antigen K2 (k, Cellano). Approximately 9% of the population has the K1 red cell phenotype, and antibodies to K1 are developed in about 5% of persons receiving a single unit of incompatible blood (Ref. 1).
Hemolytic disease of the newborn (HDN) is usually associated with maternal alloimmunization to Rh(D), but K1 incompatibilities can also cause severe hemolytic disease in newborns (Refs. 2-7). K1 sensitization from a previous pregnancy can result in HDN and complications during subsequent pregnancies if the fetus is a K1 carrier. Similar types of problems can arise in the more rare case of K2 sensitization. The identification of the fetal K1/K2 genotype would be of particular significance in situations in which the father is a K1/K2 heterozygote. Since the mother must be a homozygote in order to have been previously sensitized, there is a 50% chance that the fetus with a K:1,2 father is in danger of HDN. Because of this 50% risk, identification of the homozygosity or heterozygosity of the K1/K2 genotype of the fetus in these pregnancies becomes important in order to aid in their proper management.
Kell antigens appear to be encoded in 5 sets of antithetical paired alleles expressing high and low prevalence antigens. Thus, K1 (K) and K2 (k) are products of alleles, as are K3 (Kp.sup.a), K4 (Kp.sup.b) and K21 (Kp.sup.c); K6 (Js.sup.a) and K7 (Js.sup.b); K17 and K11; and K24 and K14. However, a number of high prevalence antigens such as K12, K13, K18 and K22 are independently expressed. These relationships, and their place in the Kell system, have been established through the years by serological analyses of informative families (Refs. 8-12). A recently developed immunological test, MAIEA, which uses monoclonal antibodies to different Kell antigens, indicates that certain of the identified antigens occur in spatially distinct regions of the glycoprotein (Ref. 13). Thus, K1/K2, and K6/K7 are close together, while K3/K4 epitope is in a different location and K18 is in yet another protein domain (Ref. 38). Kell inheritance is autosomal and codominant, and the gene for the Kell protein (KEL) has been mapped to chromosome 7q33 (Refs. 14-17).
A variety of studies, including a molecular cloning, established that Kell blood group antigens are carried on a 93 kDa type II glycoprotein (Refs. 18-24) found on the surface of red blood cells (Ref. 13). The Kell protein has a short, 46 amino acid, N-terminal domain in the cytoplasm, and a large C-terminal portion, of 665 amino acids, on the external surface of the red cell. All of the carbohydrates are N-linked (Ref. 25), probably located in 5 sites, at asparagines 93, 115, 191, 345 and 627. Early biochemical studies suggested that Kell antigens reside on a protein whose conformation is largely dependent on disulfide bonds (Ref. 26). The Kell protein has 16 cysteine residues, one in the transmembrane region and 15 in the external portion (Ref. 24). Reduction of red cells by sulfhydryl reagents results in loss of Kell antigens and exposure of some neo-epitopes (Ref. 26).
The determination of Kell genotype has heretofore been confined to methods of detecting and identifying Kell antigens. Such methods employ antibodies or other compounds which identify and interact with the Kell protein or portions thereof. For example, various antibodies specific for particular Kell antigens have been identified (Refs. 13, 22). Agglutination methods for detecting Kell protein and other blood group antigens are described in U.S. Pat. Nos. 5,324,479, 5,302,512, 5,213,963, 4,560,647, 4,403,042, 4,358,436, and 4,148,607. These methods provide information about expressed protein profiles, not about protein molecular structure or the molecular genetic makeup of the individual. Such methods provide limited information, and generally require blood samples from subjects being examined. Determining fetal Kell phenotype also normally requires a fetal blood sample. This involves a potentially dangerous procedure in which the fetus is susceptible to hemorrhage and possibly death. None of the methods previously described discloses any method by which Kell genotype might be determined.
As a result, there exists a need for a method of safely and conveniently detecting Kell genotype. It would also be desirable to provide a method for determining Kell genotype in a fetus without the requirement for obtaining blood samples. A test based on DNA samples taken from amniotic cells would allow the clinician to avoid the risk of harm to the fetus and to more accurately predict the potential of anti-K-associated HDN.